Astaxanthin Alleviates Nociceptive Pain By Regulating P38 MAPK And Nrf2/HO-1 Pathways In Mice

Background:Nociceptive pain is a series of physiological responses caused by nociceptive stimulation activating peripheral nociceptors,which can cause nerve reflex and make corresponding behavioral responses,so that the tissue can reduce the degree of injury to the lowest level.Usually the pain disappears when the injury is cured,but if the nociceptive stimulation persists,acute nociceptive pain turns into chronic nociceptive pain.In clinic,muscle,fascia,phloem inflammation and various types of osteoarthritis are important causes of chronic nociceptive pain.With the increasingly serious aging of the population and the increasing number of bad living habits of modern people,this type of pain has received more and more attention.At present,it is believed that the pathogenesis of nociceptive pain can be divided into two aspects: peripheral sensitization and central sensitization,in which peripheral sensitization plays a key role.In recent years,many studies have shown that the activation of inflammatory substances and the activation of continuous stimulation and oxidative stress play an important role in peripheral sensitization.Inflammation is a complex defense response of the human body to infection and injury.Inflammation can also promote the process of tissue repair through the role of immune cells and inflammatory mediators.However,the activation and continuous stimulation of excessive inflammatory substances are harmful to the human body and may cause or aggravate many diseases.Mitogen-activated protein kinase(MAPK)plays a key role in the development of inflammation,in which the activation of p38 MAPK is considered to be very important for the induction and maintenance of chronic inflammation,and it is also a crucial target for cytokine inhibition and antiinflammatory drug design.Oxidative stress(OS)response is caused by excessive production of intracellular reactive oxygen species(ROS)that exceeds the scavenging capacity of the antioxidant defense system in the human body,which leads to a series of adverse reactions,including glial cell activation,mitochondrial dysfunction and inflammation.The imbalance between oxidation and antioxidation is ultimately involved in the occurrence of nociceptive pain.Nuclear factor-NF-E2 related factor 2 related factor2(Nrf2)is a key transcription factor in the regulation of oxidative stress.It plays an important role in regulating a variety of antioxidant defense responses in vivo.It has been suggested that Nrf2 pathway may be involved in the pathogenesis of nociceptive pain,but the regulatory role of Nrf2 in nociceptive pain is not clear.Oral drugs are the main means to relieve nociceptive pain in clinic,and the classic representative drugs are nonsteroidal anti-inflammatory drugs.These drugs can relieve nociceptive pain by inhibiting cyclooxygenase and reducing the synthesis of prostaglandins.However,long-term oral administration of these drugs may lead to serious adverse effects such as digestive tract ulcers and increase the incidence of cardiovascular events,which limits their application in some elderly patients or patients with other serious underlying diseases.Therefore,it is imperative to find a new alternative drug.Astaxanthin(AST)is a fat-soluble orange-red carotenoid pigment produced by a variety of microorganisms and marine animals.Because of its strong anti-inflammation,anti-oxidation,anti-apoptosis and anti-aging,it is recommended as dietary supplements and functional foods.It has been pointed out that AST plays an important role in improving inflammatory diseases such as arteriosclerosis,inflammatory bowel disease,septicemia,rheumatoid arthritis,gastritis and encephalitis.The purpose of this study is to explore whether astaxanthin can exert analgesic effect on chronic nociceptive pain mouse model by regulating p38 MAPK-mediated inflammatory pathway and Nrf2-mediated oxidative stress pathway,so as to provide a new theoretical basis for the potential clinical application of AST in nociceptive pain treatment.Methods:Part I: The analgesic effect of astaxanthin in nociceptive pain miceIn order to observe the analgesic effect of AST in nociceptive pain mice induced by CFA,mice were divided into five experimental groups: saline,CFA,CFA + DMSO,CFA treated with 5 mg/kg AST,and CFA treated with 10 mg/kg AST.DMSO and AST were intraperitoneally injected once a day for 14 d,starting from day 1 after the CFA injection.Behavioral testing was performed at baseline and then on days 1,3,5,7,11,and 14.The animals were sacrificed on day 7 to collect the ipsilateral hind paw and L4-6 dorsal root ganglion(DRG)tissues for molecular studies.Additional CFA mice treated with DMSO and AST were used for immunofluorescent and immunochemical studies.Part II:(1)Astaxanthin can play an analgesic role in nociceptive pain mouse model by regulating p38 MAPK pathway.To examine the time course of changes in pain behaviors and expression of p38 MAPK in the CFA model,mice were assigned to saline and CFA groups.The CFA group received intraplantar CFA(20 μL)injection into the left hind paw,while the saline group received the same volume of saline injection.Each group was subjected to behavioral testing prior to injection(baseline)and on days 1,3,5,7,11,and 14.CFA group mice were sacrificed after behavioral measurement at each time point,and saline group mice were sacrificed at the final time point.Protein samples were extracted from the ipsilateral hind paw and L4-6 DRG of the injured side for molecular studies.To examine the role of p38 MAPK in the regulation of pain,mice were divided into four experimental groups: control,control + DMSO,control treated with 10 mg/kg p38 MAPK agonist anisomycin,and control treated with 15 mg/kg anisomycin.DMSO and anisomycin were administered with interplantar injections into the left hind paw.Behavioral testing was performed at baseline and then at 30,60,90,and 120 min.The animals were sacrificed 60 min(the most obvious time point)after injection,and their hind paw and DRG tissues were collected for molecular studies.To examine the role of p38 MAPK in the regulation of pain,mice were divided into four experimental groups: CFA,CFA + DMSO,CFA treated with 0.5 mg/kg of p38 MAPK inhibitor SB203580,and CFA treated with 1 mg/kg SB203580.DMSO and SB203580 were injected intraperitoneally.Behavioral testing was performed at baseline and then at 30,60,90,and 120 min.The animals were sacrificed 60 min(the most obvious time point)after injection,and their hind paw and DRG tissues were collected for molecular studies.To verify that AST relieves CFA-induced nociceptive pain by regulating p38 MAPK,mice were divided into five experimental groups: saline,CFA,CFA + DMSO,CFA treated with 5 mg/kg AST,and CFA treated with 10 mg/kg AST.DMSO and AST were intraperitoneally injected once a day for 7 d,starting from day 1 after the CFA injection.The animals were sacrificed on day 7 to collect the ipsilateral hind paw and L4-6 dorsal root ganglion(DRG)tissues for molecular studies.In order to observe the role of AST in nociceptive pain induced by p38 MAPK,mice were divided into four experimental groups: Control,Control+DMSO,Anisomycin(15mg/kg)+ DMSO and Anisomycin(15mg/kg)+ AST(10mg/kg).At 30 min after injection of Anisomycin(15mg/kg),DMSO and AST were injected intraperitoneally and behavioral tests were performed.Part II:(2)Astaxanthin can play an analgesic role in nociceptive pain mice by regulating Nrf2/HO-1 pathway.To examine the time course of changes in pain behaviors and expression of Nrf2,and HO-1 in the CFA model,mice were assigned to saline and CFA groups.The CFA group received intraplantar CFA(20 μL)injection into the left hind paw,while the saline group received the same volume of saline injection.Each group was subjected to behavioral testing prior to injection(baseline)and on days 1,3,5,7,11,and 14.CFA group mice were sacrificed after behavioral measurement at each time point,and saline group mice were sacrificed at the final time point.Protein samples were extracted from the ipsilateral hind paw and L4-6 DRG of the injured side for molecular studies.To examine the role of Nrf2 in the regulation of pain,mice were divided into four experimental groups: control,control + DMSO,control treated with 10 mg/kg of Nrf2 inhibitor trigonelline,and control treated with 20 mg/kg trigonelline.DMSO and trigonelline were administered with interplantar injections into the left hind paw.Behavioral testing was performed at baseline and then at 30,60,90,and 120 min.The animals were sacrificed 60 min after injection,and their hind paw and DRG tissues were collected for molecular studies.To examine the role of Nrf2 in CFA induced pain,mice were divided into four experimental groups: CFA,CFA + DMSO,CFA treated with 50 mg/kg of Nrf2 activator oltipraz,and CFA treated with 100 mg/kg oltipraz.DMSO and oltipraz were injected intraperitoneally.Behavioral testing was performed at baseline and then at 30,60,90,and120 min.The animals were sacrificed 60 min after injection,and their hind paw and DRG tissues were collected for molecular studies.To verify that AST relieves CFA-induced nociceptive pain by regulating Nrf2/HO-1,mice were divided into five experimental groups: saline,CFA,CFA + DMSO,CFA treated with 5 mg/kg AST,and CFA treated with 10 mg/kg AST.DMSO and AST were intraperitoneally injected once a day for 7 d,starting from day 1 after the CFA injection.The animals were sacrificed on day 7 to collect the ipsilateral hind paw and L4-6 dorsal root ganglion(DRG)tissues for molecular studies.To observe the role of AST in nociceptive pain induced by inhibition of Nrf2 pathway,mice were divided into four experimental groups: Control,Control+DMSO,trigonelline(20mg/kg)+ DMSO and trigonelline(20mg/kg)+ AST(10mg/kg).After injection of trigonelline(20mg/kg),30 min,DMSO and AST were injected intraperitoneally and behavioral tests were performed.Results:Part I: The analgesic effect of astaxanthin in nociceptive pain mice1.After intraperitoneal injection of different doses of AST,the paw withdrawal threshold and paw withdrawal latency of CFA mice were significantly increased.2.Intraperitoneal injection of different doses of AST significantly improved the degree of plantar swelling in CFA mice,reduced the thickness of plantar swelling and reduced the infiltration of inflammatory cells in plantar sections.On the 7th day after the establishment of CFA model,the immunofluorescence expression of macrophage marker F4-80 in plantar and dorsal root ganglion was significantly up-regulated,and the increase of F4-80 was reversed by different doses of AST treatment.3.Intraperitoneal injection of different doses of AST significantly decreased the expression of inflammatory factors IL-1 β,IL-6 and TNF-α in plantar and dorsal root ganglia of CFA mice,decreased the expression of pro-inflammatory marker i NOS,and increased the expression of anti-inflammatory marker Arg1.Part II:(1)Astaxanthin can play an analgesic role in nociceptive pain mouse model by regulating p38 MAPK pathway.1.Western blot analysis showed that compared with saline group,the expression of p-p38 MAPK in plantar and dorsal root ganglia of CFA mice increased significantly from day 1 to day 5.2.Compared with the control group and DMSO group,the paw withdrawal threshold and paw withdrawal latency of naive mice treated with different doses of p38 MAPK agonist decreased.Western blot analysis showed that 1 hour after administration of p38 MAPK agonist,the expression of p-p38 MAPK in plantar and dorsal root ganglia increased significantly.3.The paw withdrawal threshold and paw withdrawal latency of CFA mice were increased by intraperitoneal injection of different doses of p38 MAPK inhibitors.Western blot analysis showed that compared with CFA group and CFA+DMSO group,the expression of p-p38 MAPK in plantar and dorsal root ganglia decreased significantly after intraperitoneal administration of p38 MAPK inhibitor.4.Western blot analysis showed that compared with CFA and CFA+DMSO groups,the expression of p-p38 MAPK in plantar and dorsal root ganglia of CFA mice decreased significantly after intraperitoneal injection of different doses of AST.5.The paw withdrawal threshold and paw withdrawal latency of CFA mice were decreased after injection of different doses of p38 MAPK agonist.After intraperitoneal injection of different doses of AST,the above phenomena were reversed,and the paw withdrawal threshold and paw withdrawal latency were increased.Part II:(2)Astaxanthin can play an analgesic role in nociceptive pain mice by regulating Nrf2/HO-1 pathway.1.Western blot analysis showed that compared with saline group,the expression of Nrf2 and HO-1 in plantar and dorsal root ganglia of CFA mice increased,and the expression of Nrf2 decreased gradually from day 3.2.Compared with the control group and DMSO group,the paw withdrawal threshold and paw withdrawal latency of mice were decreased after plantar injection of different doses of Nrf2 inhibitor.Western blot analysis showed that the expression of Nrf2 and HO-1 in plantar and dorsal root ganglia decreased significantly after administration of Nrf2 inhibitor.3.The paw withdrawal threshold and paw withdrawal latency of CFA mice were increased by intraperitoneal injection of different doses of Nrf2 agonist.Western blot analysis showed that compared with CFA group and CFA+DMSO group,the expression of Nrf2 and HO-1 in plantar and dorsal root ganglia increased significantly after intraperitoneal administration of Nrf2 agonist.4.Western blot analysis showed that compared with CFA and CFA+DMSO groups,the expression of Nrf2 and HO-1 in plantar and dorsal root ganglia of CFA mice increased significantly after intraperitoneal injection of different doses of AST.Immunofluorescence showed that compared with CFA and CFA+DMSO groups,the expression of Nrf2 and HO-1 was significantly up-regulated after intraperitoneal injection of different doses of AST.5.The paw withdrawal threshold and paw withdrawal latency of naive mice were decreased after plantar injection of different doses of Nrf2 inhibitor.After intraperitoneal injection of different doses of AST,the above phenomena were reversed,and the paw withdrawal threshold and paw withdrawal latency increased.Conclusion1.Astaxanthin can partially alleviate the mechanical hyperalgesia and thermal hyperalgesia in CFA-induced nociceptive pain mice.Astaxanthin could inhibit the inflammatory reaction,reduced the expression of macrophages,inflammatory factors and proinflammatory marker i NOS.Astaxanthin increased the expression of antiinflammatory marker Arg1.2.p38 MAPK participates in the expression of pain in CFA mice.The activation of P38 MAPK induced mechanical hyperalgesia and thermal hyperalgesia in mice.Inhibition of P38 MAPK activation alleviates pain-like behavior changes in mice.Astaxanthin can inhibit p-p38 MAPK activation and alleviate pain-like behavior changes caused by P38 MAPK activation in mice.3.Nrf2 and HO-1 pathways are involved in the pain expression of CFA mice.Inhibition of Nrf2 pathway can induce mechanical hyperalgesia and thermal hyperalgesia in CFA mice.Activation of Nrf2 pathway can partially alleviate the pain-like behavior of CFA mice.Astaxanthin can activate Nrf2 and HO-1 pathways and alleviate the pain-like behavioral changes in mice caused by Nrf2 inhibition.